The integration of temozolomide (TMZ) chemotherapy into therapy for newly diagnosed glioblastoma multiforme (GBM) has a major impact on survival for a subset of patients. Unfortunately, essentially all patients develop resistance to TMZ, and consequently, understanding the mechanisms of TMZ resistance development is critically important. Using primary GBM xenograft models, we have demonstrated that acquired TMZ resistance can be induced by 2 distinct mechanisms within the same tumor with one mechanism being driven by increased histone acetylation within the promoter of a critical DNA repair protein MGMT. Moreover, co-treatment with the histone deacetylase inhibitor SAHA promotes development of this epigenetically-driven resistance mechanism. Whole exome-seq and RNA-seq in TMZ-resistant models without MGMT elevation identified mutations in DNA ligase IV, Ku70 and Ku80, which are key components in the non-homologous end-joining (NHEJ) DNA repair pathway. Consistent with defects in this critical pathway, these mutations were associated with delayed DNA repair kinetics and a paradoxical increase in radiation responsiveness. The focus of this application is to investigate these 2 distinct mechanisms of TMZ resistance emergence using our patient derived xenograft models and patient tumor tissues. The planned aims are: AIM 1: Define the changes in the chromatin-modifying complexes regulating MGMT upregulation. Our preliminary data suggest that distinct chromatin modifying complexes suppress MGMT expression in different MGMT-methylated GBM xenograft lines, and we hypothesize that the specific resident complex within the MGMT promoter will define the effects of epigenetically-targeted therapies on MGMT up-regulation. AIM 2: Test whether DNA damage-induced alterations in heterochromatin promote evolution of TMZ resistance. DNA damage induced by TMZ or radiation triggers a highly orchestrated DNA damage response that includes chromatin remodeling, and we hypothesize that MGMT upregulation requires focal chromatin opening within the MGMT promoter in association with damage repair AIM 3: Define whether deregulation of DNA repair contributes to mechanisms of TMZ resistance in MGMT non-expressing tumors. Based on our previous work demonstrating that suppression of NHEJ activity can abrogate the cytotoxicity of PARP inhibitors, we hypothesize that a similar disruption of NHEJ through acquired mutations contributes to TMZ resistance, while leading to an increase in radiation responsiveness. Defining the mechanism(s) of TMZ resistance associated with radiation hypersensitivity may provide a strategy for selecting patients most likely to benefit from re-irradiation following tumor progression.